The overall goal of our research is a better understanding of biological membrane structure and function, including lipid-protein and protein- protein interaction, and membrane protein folding. Development of powerful biophysical and biochemical methods, especially fluorescence techniques, is of special concern. Specifically, our goal is to understand the behavior of diphtheria toxin and related proteins. Three steps in the entry of toxin into cells are under study. First, how and why the toxin changes from a hydrophilic to hydrophobic state. Second, the conformation of membrane-inserted toxin. Third, how the poisonous A domain of the toxin is released from the membrane and enters the cytoplasm. To characterized these processes the toxin will be examined both in solution and when inserted in model membranes. A variety of methods will be used to analyze toxin structure including chemical labeling, fluorescence, proteolytic, immunological, and calorimetric approaches. Both wild type toxin and mutants containing single Trp and single sulfhydryl groups introduced by site-directed mutagenesis will be studied. This will allow site-specific labeling and determination of the location of defined sites. The behavior of whole toxin and its isolated A domain will be compared in order to define the role of individual domains in the translocation process. The project will also be extended to Pseudomonas exotoxin A, a membrane-penetrating protein that undergoes major conformational changes similar to those in diphtheria toxin. These studies should increase our general knowledge of the processes of membrane insertion and membrane translocation of proteins. They should help in understanding bacterial infection, and by revealing the similarities between toxin and viral entry into cells they should also help in understanding the viral infection process. Finally, they should help in design of therapeutically useful immunotoxins, antibody-toxin hybrids targeted to destroy specific cells (e.g. tumor cells).